Summary While many of the molecular pathways that govern mouse lung branching have been defined, little is known about early human lung branching. Our preliminary data show that the molecular and cellular mechanisms driving branching in the human fetal lung are different from those in mouse. Lung branching morphogenesis relies on several cellular and molecular events including cell migration, proliferation, proximal- distal patterning and epithelial-mesenchymal crosstalk. Proximal-distal patterning in the mouse lung is demarcated by the exclusive expression of Sox2 and Sox9 in the proximal and distal compartments respectively, and regulated by FGF signaling. Careful analyses of the human lung during the pseudoglandular stage revealed the presence of a double SOX2+/SOX9+ progenitor cell population in the distal epithelial buds, that is required for branching. Our preliminary data showed that FGF10 does not induce branching in the human fetal lung, unlike in mouse lung. In contrast, we showed that FGF18 induces branching in human lung explants, and a concomitant decrease of FGF18 and smooth muscle cells (SMCs), is associated with impaired branching. Meanwhile, we observed that SMCs extend to the periphery of the human developing lung, and seem to arrange prior to the emergence of new epithelial buds. Based on these observations, we hypothesize that FGF18, but not FGF10, plays an important role in driving human lung branching through coordinate epithelial/mesenchymal signals leading to SMC differentiation and migration, that in turn contract to mechanically guide branching morphogenesis in early human lung development. In the first aim, we will define the role of coordinate epithelial/mesenchymal FGF18 signaling in promoting early human fetal lung development. In this aim we will use complementary gain and loss of function approaches to A) determine the effect of FGF18 in the branching of human fetal lung explants, B) define whether FGF18 acts directly on the epithelium to promote branching and maintenance of SOX2/SOX9 progenitors and C) identify the effect of FGF18 signaling on mesenchymal progenitors and SMC proliferation, migration and differentiation. In the second aim, we will determine the dynamic and mechanical functions of SMCs in directing epithelial branching in human fetal lung. In this aim, we will A) determine in real time how differentiation and dynamic movement of SMCs drive epithelial branching in human lung explants in vitro, B) determine the effect of SMC contractility on human lung branching, using inhibitors of F-actin polymerization and myosin activation, and C) determine the epithelial-SMC interactions required for proper branching of the human lung. Congenital small lung (CSL), also known as pulmonary hypoplasia, is a common neonatal lung condition affecting approximately that may result from different insults affecting different developmental mechanisms. Understanding the mechanisms underlying early human lung development will transform our concepts of human lung development, and thus may allow for the discovery of possible therapeutic avenues to restore or enhance lung development for neonates with CSL.